Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

[The contraindications in blood donation. Impact of the decree of January 12, 2009]. / Les contre-indications au don du sang. Impact de l'arrêté du 12 janvier 2009.

The ministerial decree of 12 January 2009 fixing the criteria for the selection of blood donors establishes the list of contraindications for blood donation according to the appendix to the European directive 2004/33/EC. This text regroups the preexisting guidelines and reinforces their regulatory status, which illustrates the importance attributed to the selection of donors within the dispositions ensuring transfusion safety. It introduces more flexibility with respect to the preexisting criteria taking into account the improvement in life expectancy and the continual increase in transfusion requirements. Thus, the rules for donation authorize up to 24 donations annually and one may give whole blood up to six times per year for men and four times for women, up to the age of 70, the risk of anaemia now being well controlled. The medical rules are more flexible in the areas of haemochromatosis, allergies and autoimmune diseases and for the collection of plasma for fractionation. Some expected changes were not approved in certain cases due to the preeminence of the corresponding criteria in the European directive, like for cured cancers, in other cases in view of an analysis of the available epidemiological data or potential risks like for sexual relations between men and the risks linked to prions or emerging agents (transfusion antecedents). These selection criteria will be revised every year, which should permit their adaptation to the current state of knowledge, although the extent of the revisions will be limited by the updating of the European text on which they are based.

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone.

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes.

Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli-T7 expression system.

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.

[Evaluation of measurements intended to develop regularity of blood donation]. / Evaluation de mesures destinées à développer la régularité du don de sang.

An increase in the regularity of blood donation is desirable for two main reasons. First, the lower incidence of viral disease markers in regular donors when compared to first donors could help to reduce risks of transmissible diseases. Second, a higher frequency of donation could contribute to a more satisfactory supply of blood products. Two measures implemented with the aim of increasing the regularity of blood donation were evaluated: (i) an increase in the annual frequency of blood collection by mobile teams at collection sites, and (ii) a "next donation document" given to each donor indicating the earliest possible date of the next donation. The regularity index was calculated as the mean number of cellular donations (whole blood and platelet apheresis) per donor per year, over two consecutive periods: 1-7-1993-30-6-1994 (P1) and 1-7-1994-30-6-1995 (P2). The junction of these two periods corresponded to the date of introduction of the "next donation document" and to the time of reinforcement of the mobile collection frequency. First donors in either period were not taken into account in the study. A significant relationship was observed between the annual frequency of mobile collection and the average number of donations per donor (comparison test of two means on large samples, p < 0.001 in all the cases excepted those of stable mobile collection numbers). Thus, in the first period, the average donation rate was the lowest on sites having only one mobile collection per year (M: 1.55, W: 1.38) and the highest on sites with five mobile collections per year (M: 2.05, W: 1.71). These average numbers significantly increased in the second period as compared to the first (M: +4.8%, W: +1.8%; comparison test of two means from paired series, p < 0.001), as did the yearly frequency of mobile blood collections (+9.2%). On the other hand, the "next donation document" was not associated to any change in the regularity index. The results of this study therefore showed an increase in the frequency of visits of mobile collection teams to be the main factor leading to an improvement in the regularity of blood donation. Moreover, this evolution was associated with a decrease in the incidence of viral markers detected at donation.

Acyl transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids.

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with alpha-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the Km(amide) values (e.g., Km(amide) = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (kcat) were obtained with linear aliphatic amides (e.g., kcat = 333 s-1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, alpha-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., KmNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be L-enantioselective towards alpha-hydroxy- and alpha-aminoamides.

[Staff certification for mobile blood collection units]. / Une expérience de certification du personnel des prélèvements en collectes mobiles.

Training and official acknowledgment of the competence of each staff member are essential to the quality and safety of collected blood products prepared and delivered by a blood transfusion center. A procedure was created to indicate in detail the methods employed to implement such accreditation. Based on individual training according to activity, it defines for each type of activity (secretary, physician, collector, driver) the required theoretical and practical knowledge of his/her position. Accreditation, consisting of assessment of the degree of competence attained in these areas of responsibility, was applied to the members of mobile blood collection teams in 1995. No major deficiency was detected, and this certification was well accepted by the staff. In order to complete this initial accreditation, blood collection abnormalities (inadequate blood volumes, clots or defective welding of tubing) were assessed for each collector individually. Comparison of these abnormalities in qualified nurses and laboratory technicians with a blood collection diploma showed no differences. On the other hand, significantly higher numbers of abnormalities were found in intermittent as compared to regular collectors and in senior as compared to new collectors. The applied corrective measures led to obviation of differences and improvement in performance. In 1996, in the first individual evaluation of medical selection carried out by each physician, discrepancies of one to 20 donors (0.7-14.2%) were observed from one doctor to another in the frequency of elimination of candidates for blood donation after the medical interview. Regular meetings with physicians resulted in reducing these discrepancies to one to 3.1 donors (4.6-14.1%) in 1997. In conclusion, the association of an initial accreditation procedure with an individual follow-up of work quality allowed satisfactory assessment of the training and competence of staff members. This kind of method could be extended to those working in other fields of transfusion medicine.

[Quality control of homologous blood collection activities: 5 years' experience]. / Contrôle de qualité des activités de prélévements homologues: 5 ans d'expérience.

Quality control (QC) of blood collection activities for transfusion is a regulatory requirement. The authors report on their experience in this field over the past 5 years. In their institution, this QC is based on both the recording and analyzing of predefined data, as well as the search for an active collaboration from each person involved in these activities. QC of medical selection relies on the assessment of several associated criteria: effectiveness of the information given to blood donors for recruitment, preparation of the medical interview and encouragement to perform regular donations; frequency of donors deferred after the medical interview; frequency of biological abnormalities detected at donation; results of the inquiries into the corresponding medical interviews following adverse transfusion reactions. The quantitative and qualitative evaluation of blood collection permits assessment of the quality of the blood collection program, collection procedures and directly derived blood products. Quality assessment of facilities and equipment is also included in this QC. Results have been improving in recent years, especially regarding medical selection. In particular, an increase in the mean donation rate of donors, a decrease in biological abnormalities detected at donation and an absence of adverse transfusion reactions attributed after inquiry to an inadequate medical interview have been noticed. A decrease in both shortage and outdating of labile blood products likewise indicates an improvement of blood collection planning. However, this QC reveals deficiencies in the information given to donors and a lack of analysis of the data specific to first time donors. In order to further improve the efficiency of QC, these results now require comparison with similar data collected on a nation-wide scale.

[Comparative validation of manual and automated methods for mixing and volume control of total blood samples]. / Validation comparative de méthodes manuelle et automatisée pour l'agitation et le contrôle de volume des prélèvements de sang total.

During blood collection, agitation and volume limitations are critical to ensure thorough mixing of the blood with the anticoagulant and obtention of the predetermined volume. These 2 factors are essential to prevent blood activation and to obtain well standardized blood products. The objective of this study was to compare the quality of the blood collected using 2 types of collection method: tripping of a scale at a predetermined volume limit of 450 mL in the presence of manual agitation, and the 3 blood collection monitors currently available in France. A minimum of 100 collection procedures was performed for each of the 4 methods tested. Results were found to be equivalent using either the manual or the automated procedures with regard to both the accuracy and reproducibility of the blood volumes obtained and the collection times and flow rates. The characteristics of the red blood cell concentrates, platelet concentrates and plasma units prepared from the first 30 collections of each group were assessed and compared to regulatory requirements. The quality of all these products was found to be comparable to that currently observed at quality control and no product was rejected at the release control for reasons of poor collection. An assessment of the practicability of the different methods showed that the automated devices are subject to practical difficulties involving transport and battery loading. In addition, the cost of this equipment is approximately 5 times higher than that of the scales. In conclusion, the results of this study show that in our hands, no significant advantage could be expected from the use of automated blood collection monitors as compared to simple scales with manual mixing. These results further raise the question of the applicability to labile blood products of the comparative validations currently accepted in the pharmaceutical industry, in order to allow the use of correctly validated alternative methods.

[Quantitative bacteriological evaluation of a method for skin disinfection in blood donors]. / Evaluation bactériologique quantitative d'une méthode de désinfection cutanée chez les donneurs de sang.

Skin disinfection at the site of venipuncture is a critical point in every blood transfusion collection procedure, as it contributes to ensure the bacterial safety of transfusion. Quantitative and qualitative analysis of bacteria present in the antecubital fossae before and after skin disinfection may be one method of assessing the anti-bacterial efficiency of disinfection. Swab culture systems and contact plates are the two techniques usually employed for this purpose. A washing and swabbing technique was used to quantify bacteria before and skin disinfection of the antecubital fossae in blood donors. This contra-placebo study was carried out on 32 donors, each of whom served as his own control, with a random choice of test arm and opposing control arm. Bacterial counts were determined in the antecubital fossae without skin disinfection (control, n = 32) and after a 3 step skin preparation procedure (cleaning, wiping, disinfection) using placebo (distilled water, n = 16) or an antiseptic product (mixture of chlorexidine, benzalkonium chloride and benzylic alcohol, n = 16). The absence of a statistical difference in bacterial counts between the right and left antecubital fossae without disinfection was controlled in a preliminary study of 20 subjects. Mean bacterial counts were 25,000/cm2 and 27,400/cm2 respectively for aerobic and anaerobic bacteria before disinfection, with a wide variation in results between individuals. When using placebo, preparation of the venipuncture site by the 3 step method (cleaning, wiping, disinfection) resulted in a non significant mean reduction of 0.56 log in aerobic and anaerobic bacteria. Using the antiseptic product, the same method resulted in a significant mean reduction of 1.8 and 1.7 log respectively in aerobic (p = 0.015) and anaerobic flora (p = 0.005). On an average, 2,750 aerobic bacteria/cm2 and 2,910 anaerobic bacteria/cm2 remained after disinfection, while qualitative analysis showed that disinfection suppressed the transitory flora in all cases but left part of the resident flora in 12/16 cases. These findings are comparable to those of other studies carried out to evaluate this kind of technique for the disinfection of operation sites. In comparison with other techniques classically employed for this type of evaluation (swab systems or contact plates), the method used in this study was the advantage of allowing the quantification of the reduction in bacteria. Hence this method could be employed for comparative assessment of skin disinfection techniques with the aim of improving their anti-bacterial efficiency and could also make possible the definition of a minimum bacterial count (resident flora) to be obtained in all cases after disinfection.

Amide metabolism: a putative ABC transporter in Rhodococcus sp. R312.

The DNA sequence has been determined upstream of the amiE structural gene in the amidase operon of Rhodococcus sp. R312 and a new ORF (amiS2) identified. The amiS2 gene encodes a potential 206 amino acid (aa) protein containing a high proportion of hydrophobic residues. The AmiS2 protein possesses high homology to the ORFP3, amiS and ureI gene products from the Mycobacterium smegmatis (Ms) acetamidase operon, Pseudomonas aeruginosa (Pa) amidase operon and Helicobacter pylori (Hp) urease operon, respectively. Hydropathic analysis and secondary structure prediction of AmiS2 suggested the presence of seven potential transmembrane (TM) alpha-helices. Sequence analysis of the amiB2 gene, located downstream of the Rhodococcus sp. R312 amiE gene, showed that it encoded a 351-aa protein containing a potential ATP-binding motif. AmiB2 showed significant homology with the ATP-binding subunit of the bacterial Clp protease and high homology with the amiB product located within the Pa amidase operon. AmiB2 and AmiS2 appear to be two components of a recently identified novel family of ABC transporters (Wilson et al., 1995) and might be responsible for the adsorption of amidase substrates or release of their hydrolysis products.

Study of the amidase signature group.

Computer methods for database search, multiple alignment and cluster analysis indicated significant homology between amino-acid sequences of 21 amidases or amidohydrolases (EC 3.5). All of them were found to be involved in the reduction of organic nitrogen compounds and ammonia production. A conserved motif was found which may be important in amide binding and in catalytic mechanisms. Homology studies between these amidases and some ureases, nitrilases and acyl-transferases or enzymes with unknown functions provided new insight into the evolution of these proteins. Dissemination of these genes seemed to be facilitated by transfer of genetic elements such as transposons and plasmids.

Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation.

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.

Karyotype studies on different strains of Candida molischiana by pulsed-field gel electrophoresis.

We used pulsed-field gel electrophoresis to compare the electrophoretic karyotype of a Candida molischiana mutant, de-repressed for beta-glucosidase production, with a wild-type strain and a reference strain. The chromosomal organization in this mutant yeast was found to be quite different. Hybridization patterns and the relative fluorescence of all bands indicated eight chromosomes in the mutant strain and seven in the other two. All three strains seemed to be haploid, with an estimated genome size of 12 Mb; the beta-glucosidase gene was on the same chromosome in all of them and the SfiI restriction patterns of this chromosome indicated that it is not affected by the mutation.

Brevibacterium linens pBL33 and Rhodococcus rhodochrous pRC1 cryptic plasmids replicate in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312).

The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312). The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp. R312. They all carry the Tn903 neomycin-resistance-encoding gene (aphI).

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli.

The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.

Application of high-performance liquid chromatography to the study of the biological transformation of adiponitrile.

A procedure for the assay of nitrile hydratase and amidase activity by high-performance liquid chromatography is described. The method can be used to assay the intermediate compounds resulting from the hydrolysis of adiponitrile into adipic acid, and to determine the kinetics of the hydrolysis of these compounds using whole cells and enzyme extracts. The precision of the method makes it suitable for the determination of the enzyme parameters: Km and Vm (nitrile hydratase and amidase). Using cyanovaleramide as substrate, Km and Vm were respectively 370 mM and 2060 U/mg for nitrile hydratase and 6.6 mM and 33 U/mg for amidase.

The N-terminal amino acid sequences of Brevibacterium sp. R312 nitrile hydratase.

Nitrile hydratase from Brevibacterium sp. R312 was purified to homogeneity. The isoelectric point was 5.75. The two kinds of subunits were separated by reverse phase HPLC and their N-terminal amino acid sequences were found to be identical to those of Rhodococcus sp. N-774 nitrile hydratase.